Importance of Scaffold Flexibility/Rigidity in the Design and Directed Evolution of Artificial Metallo-β-Lactamases
We describe the design and evolution of catalytic hydrolase activity on a supramolecular protein scaffold, Zn4:C96RIDC14, which was constructed from cytochrome cb562 building blocks via a metal-templating strategy. Previously, we reported that Zn4:C96RIDC14 could be tailored with tripodal (His/His/Glu), unsaturated Zn coordination motifs in its interfaces to generate a variant termed Zn8:A104AB34, which in turn displayed catalytic activity for the hydrolysis of activated esters and β-lactam antibiotics. Zn8:A104AB34 was subsequently subjected to directed evolution via an in vivo selection strategy, leading to a variant Zn8:A104/G57AB34 which displayed enzyme-like Michaelis–Menten behavior for ampicillin hydrolysis. A criterion for the evolutionary utility or designability of a new protein structure is its ability to accommodate different active sites. With this in mind, we examined whether Zn4:C96RIDC14 could be tailored with alternative Zn coordination sites that could similarly display evolvable catalytic activities. We report here a detailed structural and functional characterization of new variant Zn8:AB54, which houses similar, unsaturated Zn coordination sites to those in Zn8:A104/G57AB34, but in completely different microenvironments. Zn8:AB54 displays Michaelis–Menten behavior for ampicillin hydrolysis without any optimization. Yet, the subsequent directed evolution of Zn8:AB54 revealed limited catalytic improvement, which we ascribed to the local protein rigidity surrounding the Zn centers and the lack of evolvable loop structures nearby. The relaxation of local rigidity via the elimination of adjacent disulfide linkages led to a considerable structural transformation with a concomitant improvement in β-lactamase activity. Our findings reaffirm previous observations that the delicate balance between protein flexibility and stability is crucial for enzyme design and evolution.
Host protein: Zn8:AB54Anchoring strategy: DativeOptimization: GeneticNotes: Supramolecular protein scaffold constructed from cytochrome cb562 building blocks, Ampicillin hydrolysis: kcat/KM = 130 min-1 * M-1
Host protein: Zn8:AB54 (mutant C96T)Anchoring strategy: DativeOptimization: GeneticNotes: Supramolecular protein scaffold constructed from cytochrome cb562 building blocks, Ampicillin hydrolysis: kcat/KM = 210 min-1 * M-1
Multifunctional Nanoenzymes from Carbonic Anhydrase Skeleton
Carbonic anhydrase (carbonic dehydratase) (CA) is a metalloenzyme that contains zinc (Zn2+) ion in its active site. CA catalyzes the reversible conversion of carbon dioxide and water to bicarbonate and protons. Zn2+ ions, which are present in the active site of the enzyme, interact with the substrate molecules directly and cause catalytic effect. In this study, a nano-enzyme system was designed in aqueous solutions at room temperature and under nitrogen atmosphere to use the CA enzyme without any pre-treatment and deformation in its structure. The novel concept ANADOLUCA (AmiNo Acid (monomer) Decorated and Light Underpinning Conjugation Approach) was used for this process, nano CA enzyme of size 93 nm was synthesized. The activity of the synthesized nano CA was measured following the change in absorbance during the conversion of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion at 348 nm for a period of 10 min at 25 °C compared with free CA enzyme. Km and Vmax values for nano CA enzyme were found to be 0.442 mM and 1.6 × 10−3 mM min-1, respectively, whereas Km and Vmax values for free CA were found to be 0.471 mM and 1.5 × 10−3 mM min-1, respectively. In addition to these, the Zn2+ ion present in the active site of the nano CA enzyme was replaced by rodium metal. This nanorodium-substituted CA has been investigated as a new reductase enzyme for the stereoselective hydrogenation of olefins. Then, the Zn2+ ion in the active site of the nano CA enzyme was replaced with manganese metal to enhance the enzyme structure, thereby gaining characteristics of peroxidase. This newly synthesized nano manganese-substituted CA enzyme was investigated for its role as a peroxidase, which could be an alternative for hydrogen peroxidases.
Max TON: ---ee: ---PDB: ---Notes: Cross-linked carbonic anhydrase nano-enzyme particles (93 nm in diameter). Hydrolysis of 4-nitrophenyl acetate.
Metal: RhReaction: HydrationMax TON: ---ee: ---PDB: ---Notes: Cross-linked carbonic anhydrase nano-enzyme particles (93 nm in diameter). Hydration of styrene.